抑制神经降压素受体1通过let-7a-3p/Bcl-w轴在胶质母细胞瘤中诱导内源性凋亡。
Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.
作者信息
Dong Zhen, Lei Qian, Yang Rui, Zhu Shunqin, Ke Xiao-Xue, Yang Liqun, Cui Hongjuan, Yi Liang
机构信息
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China.
Department of Neurosurgery, Daping Hospital, the Third Military Medical University, Chongqing 400042, China.
出版信息
Br J Cancer. 2017 Jun 6;116(12):1572-1584. doi: 10.1038/bjc.2017.126. Epub 2017 May 11.
UNLABELLED
Backgroud:Glioblastoma is a kind of highly malignant and aggressive tumours in the central nervous system. Previously, we found that neurotensin (NTS) and its high-affinity receptor 1 (NTSR1) had essential roles in cell proliferation and invasiveness of glioblastoma. Unexpectedly, cell death also appeared by inhibition of NTSR1 except for cell cycle arrest. However, the mechanisms were remained to be further explored.
METHODS
Cells treated with SR48692, a selective antagonist of NTSR1, or NTSR1 shRNA were stained with Annexin V-FITC/PI and the apoptosis was assessed by flow cytometry. Cytochrome c release was detected by using immunofluorescence. Mitochondrial membrane potential (MMP, ΔΨm) loss was stained by JC-1 and detected by immunofluorescence or flow cytometry. Apoptosis antibody array and microRNA microarray were performed to seek the potential regulators of NTSR1 inhibition-induced apoptosis. Interaction between let-7a-3p and Bcl-w 3'UTR was evaluated by using luciferase assay.
RESULTS
SR48692 induced massive apoptosis, which was related to mitochondrial cytochrome c release and MMP loss. Knockdown of NTSR1 induced slight apoptosis and significant MMP loss. In addition, NTSR1 inhibition sensitised glioblastoma cells to actinomycin D or doxorubicin-induced apoptosis. Consistently, NTSR1 inhibition-induced mitochondrial apoptosis was accompanied by downregulation of Bcl-w and Bcl-2. Restoration of Bcl-w partly rescued NTSR1 deficiency-induced apoptosis. In addition, NTSR1 deficiency promoted higher let-7a-3p expression and inhibition let-7a-3p partly rescued NTSR1 inhibition-induced apoptosis. In addition, let-7a-3p inhibition promoted 3'UTR activities of Bcl-w and the expression of c-Myc and LIN28, which were the upstream of let-7a-3p, decreased after NTSR1 inhibition.
CONCLUSIONS
NTSR1 had an important role in protecting glioblastoma from intrinsic apoptosis via c-Myc/LIN28/let-7a-3p/Bcl-w axis.
未标记
背景：胶质母细胞瘤是中枢神经系统中一种高度恶性且侵袭性强的肿瘤。此前，我们发现神经降压素（NTS）及其高亲和力受体1（NTSR1）在胶质母细胞瘤的细胞增殖和侵袭中起重要作用。出乎意料的是，除了细胞周期停滞外，抑制NTSR1还会导致细胞死亡。然而，其机制仍有待进一步探索。
方法
用NTSR1的选择性拮抗剂SR48692或NTSR1 shRNA处理细胞，用膜联蛋白V-FITC/PI染色，通过流式细胞术评估细胞凋亡。使用免疫荧光检测细胞色素c的释放。用JC-1对线粒体膜电位（MMP，ΔΨm）丧失进行染色，并通过免疫荧光或流式细胞术进行检测。进行凋亡抗体阵列和微小RNA微阵列以寻找NTSR1抑制诱导凋亡的潜在调节因子。使用荧光素酶测定评估let-7a-3p与Bcl-w 3'UTR之间的相互作用。
结果
SR48692诱导大量细胞凋亡，这与线粒体细胞色素c释放和MMP丧失有关。敲低NTSR1诱导轻微细胞凋亡和显著的MMP丧失。此外，抑制NTSR1使胶质母细胞瘤细胞对放线菌素D或阿霉素诱导的细胞凋亡敏感。一致地，NTSR1抑制诱导的线粒体凋亡伴随着Bcl-w和Bcl-2的下调。恢复Bcl-w部分挽救了NTSR1缺陷诱导的细胞凋亡。此外，NTSR1缺陷促进更高的let-7a-3p表达，抑制let-7a-3p部分挽救了NTSR1抑制诱导的细胞凋亡。此外，抑制let-7a-3p促进了Bcl-w的3'UTR活性，并且NTSR1抑制后，let-7a-3p上游的c-Myc和LIN28的表达降低。
结论
NTSR1通过c-Myc/LIN28/let-7a-3p/Bcl-w轴在保护胶质母细胞瘤免受内源性凋亡方面起重要作用。
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